The long-term objective of this application is to understand the molecular mechanisms by which Trypanosoma cruzi and Trypanosoma brucei are able to survive within the mammalian host and cause such profound disease. These flagellated protozoans have a complex life cycle, with programmed cellular differentiation regulating many aspects of parasite biology as the parasite transitions from the bloodstream of the mammalian host to the insect vector and back. This research focuses on the regulation and function of stage-specific parasite responses, with special emphasis on those enabling parasite survival and disease pathogenesis in the mammalian host. Specifically, this proposal investigates a major class of calcium signaling proteins in trypanosomes. The calflagins of T. brucei are studied in this proposal, because of genetic advancements in this organism enabling targeted gene inhibition by RNA interference. However, the conclusions derived are very likely to apply to the T. cruzi homologue as well (FCaBP), since they share >60% sequence identity, the same calcium-binding characteristics, lipid modifications, cellular localization, and lipid raft association. My preliminary work indicates that the calflagins (i) contribute to parasite virulence in murine models of infection, and (ii) are required for the differentiation from infectious forms of the parasite to transmissable forms. The first aim of this proposal seeks to identify the molecular pathway regulated by calflagin calcium-binding, through a combination of biochemical methods for purifying and characterizing interacting proteins and cellular assays for testing for deficits of calflagin mutants in hypothesized functions. The second aim seeks to determine the mechanism underlying the virulence and transmission deficit of calfalgin mutants. Because an interaction with host humoral immunity is suspected to underlie the virulence deficit of calfalgin mutants, the antibody response will be compared for calflagin RNAi and wild-type infections, and a restoration of normal virulence for calflagin mutants will be tested in B cell- deficient muMT knockout mice and in a setting of mixed co-infection with wild-type parasites. PUBLIC HEALTH RELEVANCE: These investigations have clear and direct implications to public health, as T. cruzi infects an estimated 8-11 million people in Latin America and another 100,000 in the United States. This research focuses on the ways in which these parasites are able to survive and cause disease in mammals. The proteins investigated in the proposed work, as well as interacting proteins identified by these experiments, are promising targets for drug development.